Purification, molecular characterization and ligand binding properties of the major donkey seminal plasma protein DSP-1.

Fibronectin type-II (FnII) family proteins are the major proteins in many mammalian species including bull, horse and pig. In the present study, a major FnII protein has been identified and isolated from donkey (Equus hemionus) seminal plasma, which we refer to as Donkey Seminal Plasma protein-1 (DSP-1). The amino acid sequence determined by mass spectrometry and computational modeling studies revealed that DSP-1 is homologous to other mammalian seminal plasma proteins, including bovine PDC-109 (also known as BSP-A1/A2) and equine HSP-1/2. High-resolution LC-MS analysis indicated that the protein is heterogeneously glycosylated and also contains multiple acetylations, occurring in the attached glycans. Structural and thermal stability studies on DSP-1 employing CD spectroscopy and differential scanning calorimetry showed that the protein unfolds at ~43 °C and binding to phosphorylcholine (PrC) - the head group moiety of choline phospholipids - increases its thermal stability. Intrinsic fluorescence titrations revealed that DSP-1 recognizes lyso-phosphatidylcholine with over 100-fold higher affinity than PrC. Further, interaction of DSP-1 with erythrocytes, a model cell membrane, revealed that DSP-1 binding is mediated by a specific interaction with choline phospholipids and results in membrane perturbation, suggesting that binding of this protein to sperm plasma membrane could be physiologically significant.

Structure of a mammalian sperm cation channel complex.

The cation channel of sperm (CatSper) is essential for sperm motility and fertility1,2. CatSper comprises the pore-forming proteins CATSPER1-4 and multiple auxiliary subunits, including CATSPERß, γ, δ, ε, ζ, and EFCAB91,3-9. Here we report the cryo-electron microscopy (cryo-EM) structure of the CatSper complex isolated from mouse sperm. In the extracellular view, CATSPER1-4 conform to the conventional domain-swapped voltage-gated ion channel fold10, following a counterclockwise arrangement. The auxiliary subunits CATSPERß, γ, δ and ε-each of which contains a single transmembrane segment and a large extracellular domain-constitute a pavilion-like structure that stabilizes the entire complex through interactions with CATSPER4, 1, 3 and 2, respectively. Our EM map reveals several previously uncharacterized components, exemplified by the organic anion transporter SLCO6C1. We name this channel-transporter ultracomplex the CatSpermasome. The assembly and organization details of the CatSpermasome presented here lay the foundation for the development of CatSpermasome-related treatments for male infertility and non-hormonal contraceptives.

Characterization and biological role of cysteine-rich venom protein belonging to CRISPs from turkey seminal plasma†.

Turkey semen contains cysteine-rich secretory proteins (CRISPs) that belong to the dominant seminal plasma proteins. We aimed to isolate and characterize CRISP from turkey seminal plasma and evaluate its possible involvement in yellow semen syndrome (YSS). YSS, which is well characterized, causes reduced fertility and hatchability. The protein was purified using hydrophobic interaction, gel filtration, and reverse phase chromatography. It then was subjected to identification by mass spectrometry, analysis of physicochemical properties, and specific antibody production. The biological function of the isolated protein was tested and included its effects on sperm motility and migration and sperm-egg interactions. Sperm motility was measured with the CASA system using Hobson Sperm Tracker. The reproductive tract of turkey toms was analyzed for gene expression; immunohistochemistry was used for protein localization in the male reproductive tract, spermatozoa, and inner perivitelline layer. The isolated protein was identified as cysteine-rich venom protein-like isoform X2 (CRVP X2; XP_010706464.1) and contained feature motifs of CRISP family proteins. Turkey CRVP X2 was present in both spermatozoa and seminal plasma. The extensive secretion of CRVP X2 by the epithelial cells of the epididymis and ductus deferens suggests its involvement in post-testicular sperm maturation. The internally localized CRVP X2 in the proximal part of the sperm tail might be responsible for stimulation of sperm motility. CRVP X2 on the sperm head might be involved in several events prior to fusion and may also participate in gamete fusion itself. Although the mechanisms by which CRVP X2 mediates fertilization are still unknown, the involvement of complementary sites cannot be excluded. The disturbance of CRVP X2 expression can serve as an etiologic factor of YSS in the turkey. This study expands the understanding of the detailed mechanism of fertilization in birds by clarifying the specific role of CRVP X2.

Seminal plasma AnnexinA2 protein is a relevant biomarker for stallions which require removal of seminal plasma for sperm survival upon refrigeration†.

Some stallions yield ejaculates that do not tolerate conservation by refrigeration prior to artificial insemination (AI), showing improvement after removal of most of the seminal plasma (SP) by centrifugation. In this study, the SP-proteome of 10 different stallions was defined through high-performance liquid chromatography with tandem mass spectrometry and bioinformatic analysis in relation to the ability of the ejaculates to maintain semen quality when cooled and stored at 5°C. Stallions were classified into three groups, depending on this ability: those maintaining good quality after direct extension in a commercial extender (good), stallions requiring removal of seminal plasma (RSP) to maintain seminal quality (good-RSP), and stallions, unable to maintain good semen quality even after RSP (poor). Pathway enrichment analysis of the proteins identified in whole equine SP using human orthologs was performed using g: profiler showing enriched Reactome and the Kyoto Encyclopedia of Genes and Genomes pathways related to hexose metabolism, vesicle mediated transport, post translational modification of proteins and immune response. Specific proteins overrepresented in stallions tolerating conservation by refrigeration included a peroxiredoxin-6 like protein, and transcobalamin-2, a primary vitamin B12-binding, and transport protein. Also, the protein involved in protein glycosylation, ST3 beta-galactoside alpha-2,3-sialyltransferase 1 was present in good stallions. These proteins were nearly absent in poor stallions. Particularly, annexinA2 appeared as to be the most powerful discriminant variable for identification of stallions needing RSP prior to refrigeration, with a P = 0.002 and a q value = 0.005. Overall this is the first detailed study of the equine SP-proteome, showing the potential value of specific proteins as discriminant bio-markers for clinical classification of stallions for AI.

The functions of CAP superfamily proteins in mammalian fertility and disease.

BACKGROUND: Members of the cysteine-rich secretory proteins (CRISPS), antigen 5 (Ag5) and pathogenesis-related 1 (Pr-1) (CAP) superfamily of proteins are found across the bacterial, fungal, plant and animal kingdoms. Although many CAP superfamily proteins remain poorly characterized, over the past decade evidence has accumulated, which provides insights into the functional roles of these proteins in various processes, including fertilization, immune defence and subversion, pathogen virulence, venom toxicology and cancer biology. OBJECTIVE AND RATIONALE: The aim of this article is to summarize the current state of knowledge on CAP superfamily proteins in mammalian fertility, organismal homeostasis and disease pathogenesis. SEARCH METHODS: The scientific literature search was undertaken via PubMed database on all articles published prior to November 2019. Search terms were based on following keywords: 'CAP superfamily', 'CRISP', 'Cysteine-rich secretory proteins', 'Antigen 5', 'Pathogenesis-related 1', 'male fertility', 'CAP and CTL domain containing', 'CRISPLD1', 'CRISPLD2', 'bacterial SCP', 'ion channel regulator', 'CatSper', 'PI15', 'PI16', 'CLEC', 'PRY proteins', 'ASP proteins', 'spermatogenesis', 'epididymal maturation', 'capacitation' and 'snake CRISP'. In addition to that, reference lists of primary and review article were reviewed for additional relevant publications. OUTCOMES: In this review, we discuss the breadth of knowledge on CAP superfamily proteins with regards to their protein structure, biological functions and emerging significance in reproduction, health and disease. We discuss the evolution of CAP superfamily proteins from their otherwise unembellished prokaryotic predecessors into the multi-domain and neofunctionalized members found in eukaryotic organisms today. At least in part because of the rapid evolution of these proteins, many inconsistencies in nomenclature exist within the literature. As such, and in part through the use of a maximum likelihood phylogenetic analysis of the vertebrate CRISP subfamily, we have attempted to clarify this confusion, thus allowing for a comparison of orthologous protein function between species. This framework also allows the prediction of functional relevance between species based on sequence and structural conservation. WIDER IMPLICATIONS: This review generates a picture of critical roles for CAP proteins in ion channel regulation, sterol and lipid binding and protease inhibition, and as ligands involved in the induction of multiple cellular processes.

Recombinant peptide reverses cryo-capacitation in ram sperm and improves in vitro fertilization.

Semen cryopreservation is a very important technique for assisted reproduction; however, the cryopreservation process is harmful because it results in a reduction in sperm motility and viability, and leads to premature signals of capacitation, resulting in lesser than desirable fertility rates after artificial insemination. A fraction of seminal plasma, enriched in proteins that contain type II fibronectin domains (FNII) can reverse molecular indicators of cryo-capacitation. The beneficial effects of these proteins, however, depend on the relative abundance in seminal plasma. To create a safe additive for improving frozen sperm functionality, in the present study there was cloning and expression of a recombinant peptide containing four FNII domains (named TrxA-FNIIx4-His6) and evaluation of its effect after addition to frozen/thawed ram sperm. The cDNA for this protein was expressed in E. coli and after denaturation and re-naturalization of the protein, toxicity and binding capacity were assessed. By fluorescent labelling assessment, there was binding of the protein to the thawed sperm. At the two doses used (0.15 and 0.3 µM), TrxA-FNIIx4-His6 had the capacity to reverse the molecular indicators of cryo-capacitation as indicated by the reduction on phosphorylated substrates of PKA. Furthermore, the supplementation with this protein resulted in a normal capacitation process as evidenced by the increase in the in vitro fertilization rate when the greatest concentration of the protein was evaluated (73.25 ±â€¯2.95; 40.13 ±â€¯11.82 for 0.3 µM and control, respectively). There was no effect of protein supplementation on sperm objective motility compared to untreated sperm. In conclusion, the use of TrxA-FNIIx4-His6 is a promising biotechnological approach for cryopreserving ram sperm and maintaining sperm viability.

ADS-J1 disaggregates semen-derived amyloid fibrils.

Semen-derived amyloid fibrils, comprising SEVI (semen-derived enhancer of viral infection) fibrils and SEM1 fibrils, could remarkably enhance HIV-1 sexual transmission and thus are potential targets for the development of an effective microbicide. Previously, we found that ADS-J1, apart from being an HIV-1 entry inhibitor, could also potently inhibit seminal amyloid fibrillization and block fibril-mediated enhancement of viral infection. However, the remodeling effects of ADS-J1 on mature seminal fibrils were unexplored. Herein, we investigated the capacity of ADS-J1 to disassemble seminal fibrils and the potential mode of action by applying several biophysical and biochemical measurements, combined with molecular dynamic (MD) simulations. We found that ADS-J1 effectively remodeled SEVI, SEM186-107 fibrils and endogenous seminal fibrils. Unlike epigallocatechin gallate (EGCG), a universal amyloid fibril breaker, ADS-J1 disaggregated SEVI fibrils into monomeric peptides, which was independent of oxidation reaction. MD simulations revealed that ADS-J1 displayed strong binding potency to the full-length PAP248-286 via electrostatic interactions, hydrophobic interactions and hydrogen bonds. ADS-J1 might initially bind to the fibrillar surface and then occupy the amyloid core, which eventually lead to fibril disassembly. Furthermore, the binding of ADS-J1 with PAP248-286 might induce conformational changes of PAP248-286 Disassembled PAP248-286 might not be favorable to re-aggregate into fibrils. ADS-J1 also exerts abilities to remodel a panel of amyloid fibrils, including Aß1-42, hIAPP1-37 and EP2 fibrils. ADS-J1 displays promising potential to be a combination microbicide and an effective lead-product to treat amyloidogenic diseases.

Structure, function and antagonism of semen amyloids.

Amyloid fibrils are linear polypeptide aggregates with a cross-ß structure. These fibrils are best known for their association with neurodegenerative diseases, such as Alzheimer's or Parkinson's, but they may also be used by living organisms as functional units, e.g. in the synthesis of melanin or in the formation of bacterial biofilms. About a decade ago, in a search for semen factors that modulate infection by HIV-1 (a sexually transmitted virus and the causative agent of the acquired immune deficiency syndrome (AIDS)), it was demonstrated that semen harbors amyloid fibrils capable of markedly increasing HIV infection rates. This discovery not only created novel opportunities to prevent sexual HIV-1 transmission but also stimulated research to unravel the natural role of these factors. We discuss here the identification of these intriguing structures, their molecular properties, and their effects on both sexually transmitted diseases and reproductive health. Moreover, we review strategies to antagonize semen amyloid to prevent sexual transmission of viruses.

Binder of Sperm Proteins protect ram spermatozoa from freeze-thaw damage.

Cryopreservation causes sub-lethal damage which limits the fertility of frozen thawed spermatozoa. Seminal plasma has been investigated as a cryoprotectant, but has yielded inconsistent results due to considerable variation in its constituents. Individual seminal plasma proteins offer an ideal alternative to whole seminal plasma, and several have been correlated with freezing success. Binder of Sperm Proteins (BSPs) are abundant ram seminal plasma proteins which have been suggested to have significant protective effects on ram spermatozoa during cold shock. This is in direct opposition to bull spermatozoa, where BSPs cause sperm deterioration during in vitro handling. We investigated the potential of BSP1 and BSP5 to prevent freezing associated damage to important functional parameters of ram spermatozoa. BSPs purified by size exclusion chromatography improved post thaw motility and penetration through artificial mucus. Highly purified BSP1 and BSP5, isolated by gelatin affinity and RP-HPLC, improved motility and membrane integrity, and reduced post thaw protein tyrosine phosphorylation. Exposure to BSP5 before freezing increased the amount of phosphatidylethanolamine on the sperm surface after thawing. Neither BSP1 nor BSP5 prevented freezing associated changes in membrane lipid disorder. These results suggest that BSPs may significantly improve freezing outcomes of ram spermatozoa.

Intramembranal disulfide cross-linking elucidates the super-quaternary structure of mammalian CatSpers.

CatSper is a voltage-dependent calcium channel located in the plasma membrane of the sperm flagellum and is responsible for triggering hyperactive motility. A homology model for the transmembrane region was built in which the arrangement of the subunits around the pseudo-four-fold symmetry axis was deduced by the pairing of conserved transmembranal cysteines across mammals. Directly emergent of the predicted quaternary structure is an architecture in which tetramers polymerize through additional, highly conserved cysteines, creating one or more double-rows channels extending the length of the principal piece of the mammalian sperm tail. The few species that are missing these cysteines are eusocial or otherwise monogamous, suggesting that sperm competition is selective for a disulfide-crosslinked macromolecular architecture. The model suggests testable hypotheses for how CatSper channel opening might behave in response to pH, 2-arachidonoylglycerol, and mechanical force. A flippase function is hypothesized, and a source of the concomitant disulfide isomerase activity is found in CatSper-associated proteins ß, δ and ε.

Purification, structural and biophysical characterisation of the major seminal plasma protein from Texel rams.

Spermadhesins are a group of low molecular weight proteins present in seminal plasma. In Texel rams, they represent more than 70% of the seminal plasma proteins. Although their functions have not yet been fully clarified, there is much discussion about the role of these proteins in maintaining sperm viability during and after the semen freezing process. This work sought to isolate the major component of the seminal plasma from rams of the Texel breed (O. aries SPD2) and to evaluate its structural and biophysical characteristics in order to better understand its role in spermatic viability. The protein was isolated by centrifugation and ion exchange chromatography and its biophysical properties were evaluated by circular dichroism spectrometry. Molecular dynamics simulations of the modelled protein compared to the homologous bovine protein were also carried out. The results showed that O. aries SPD2 has a transition temperature (Tm) of 65 °C and a ΔHm of 322.5 kJ mol-1, similar to the results for other spermadhesins described in the literature. The estimated composition of the secondary structure elements for the native protein is in agreement with that observed for the theoretical model. Its structural characteristics were preserved in simulations at temperatures of 27 °C and 40 °C, as was the case for bull spermadhesin. Taken together, these results suggest that the major component of the spermadhesins of Texel rams (O. aries SPD2) may play an important role in maintaining the viability of spermatozoa in fresh semen as well as after thawing.

Wbp2nl has a developmental role in establishing neural and non-neural ectodermal fates.

In many animals, maternally synthesized mRNAs are critical for primary germ layer formation. In Xenopus, several maternal mRNAs are enriched in the animal blastomere progenitors of the embryonic ectoderm. We previously identified one of these, WW-domain binding protein 2 N-terminal like (wbp2nl), that others previously characterized as a sperm protein (PAWP) that promotes meiotic resumption. Herein we demonstrate that it has an additional developmental role in regionalizing the embryonic ectoderm. Knock-down of Wbp2nl in the dorsal ectoderm reduced cranial placode and neural crest gene expression domains and expanded neural plate domains; knock-down in ventral ectoderm reduced epidermal gene expression. Conversely, increasing levels of Wbp2nl in the neural plate induced ectopic epidermal and neural crest gene expression and repressed many neural plate and cranial placode genes. The effects in the neural plate appear to be mediated, at least in part, by down-regulating chd, a BMP antagonist. Because the cellular function of Wbp2nl is not known, we mutated several predicted motifs. Expressing mutated proteins in embryos showed that a putative phosphorylation site at Thr45 and an α-helix in the PH-G domain are required to ectopically induce epidermal and neural crest genes in the neural plate. An intact YAP-binding motif also is required for ectopic epidermal gene expression as well as for down-regulating chd. This work reveals novel developmental roles for a cytoplasmic protein that promotes epidermal and neural crest formation at the expense of neural ectoderm.

Glycosidases in porcine follicular fluid and their effect on zona pellucida-AWN 1 spermadhesin interaction.

Oligosaccharide moieties on the surface of the oocyte belong to the key molecules that direct the course of fertilization and are subjected to changes during oocyte maturation in the follicle. In our study, we focused on the activities of five glycosidases in the fluids from porcine secondary and preovulatory follicles (α-l-fucosidase, α-d-galactosidase, ß-d-galactosidase, ß-D-N-acetylhexosaminidase, and α-d-mannosidase). All of them were detected active at neutral and acidic pH. However, changes in their activities associated with follicle development were observed only in the case of α-d-mannosidase, which was increased (P < 0.001), and ß-d-galactosidase, which was decreased (P < 0.001) at neutral pH, and of α-d-galactosidase and ß-N-acetylhexosaminidase, which were decreased (P < 0.0001) at the acidic pH. The comparison of glycosidases from follicular fluid and from blood plasma using red native electrophoresis revealed that most of the glycosidases are present in more than one isoenzyme form; some of them were detected mainly in the follicular fluid. Finally, we tested the effect of glycosidases on the interaction between zona pellucida and AWN 1 spermadhesin (putative sperm receptor of zona pellucida) and demonstrated that the effect of both ß-d-galactosidase and to a lesser degree α-d-mannosidase led to a decrease in this interaction. We can hypothesize that these two glycosidases modulate the amount of zona pellucida oligosaccharide moieties and/or their structures for an optimal sperm binding in pigs.

SL15: A seminal plasma-derived lectin from the sperm of llama (Lama glama).

The oviductal sperm reservoir of South American camelids is formed when sperm bind to N-acetylgalactosamine (GalNAc) on the surface of oviductal epithelium. The aim of this study was to characterize the GalNAc-binding proteins on llama sperm, and to establish their origin. Sperm-adsorbed proteins were extracted with 0.5 M KCl in Hepes-balanced salts. Sperm-adsorbed and seminal plasma proteins were then subjected to ligand blotting for their GalNAc affinity, and the labeled bands were identified by mass spectrometry. Three proteins were identified in seminal plasma versus only one in the sperm-adsorbed population; SL15, a seminal lectin, was common to both. SL15 is a homologue of Zymogen granule protein 16, homolog B-like, which belongs to the Jacalin-related lectin family. This lectin is likely presented to sperm via seminal plasma since epididymal sperm are not capable of binding GalNAc, whereas ejaculated sperm does, and its transcript was enriched predominantly in the prostate and bulbourethral glands. This is the first report of a seminal lectin in South American camelids that originates in the male reproductive tract, and is probably involved in sperm reservoir formation.

Differential modulation of the chaperone-like activity of HSP-1/2, a major protein of horse seminal plasma by anionic and cationic surfactants.

The major protein of equine seminal plasma, HSP-1/2 exhibits chaperone-like activity (CLA) by protecting various target proteins against thermal, chemical and oxidative stress. Polydispersity and surface hydrophobicity of HSP-1/2 were found to be important for its CLA. Surfactants are known to alter certain properties of proteins, e.g. hydrophobicity, charge and conformation either by altering properties of the medium or by direct binding. In the current study, thermal aggregation of alcohol dehydrogenase (ADH) and enolase has been studied in the presence of HSP-1/2, different surfactants and their combinations. The results obtained show that anionic surfactants (SDS, sodium dodecyl benzene sulfate) and neutral surfactants (tween-20, triton X-100) increase the CLA of HSP-1/2 and also inhibit aggregation of the target proteins independently. On the other hand, cationic surfactants (CTAB, alanine palmityl ester) increased the thermal aggregation of ADH and enolase and also decreased the CLA of HSP-1/2. These results are of significant interest as they show that surfactants such as SDS and tween-20 can potentially be used as anti-aggregation agents to prevent thermal aggregation of target proteins.

Structural, Functional and Phylogenetic Analysis of Sperm Lysozyme-Like Proteins.

Sperm lysozyme-like proteins belonging to c-type lysozyme family evolved in multiple forms. Lysozyme-like proteins, viz., LYZL2, LYZL3 or SLLP1, LYZL4, LYZL5 and LYZL6 are expressed in the testis of mammals. Not all members of LYZL family have been uniformly and unambiguously identified in the genome and proteome of mammals. Some studies suggested a role of SLLP1 and LYZL4 in fertilization; however, the function of other LYZL proteins is unknown. We identified all known forms of LYZL proteins in buffalo sperm by LC-MS/MS. Cloning and sequence analysis of the Lyzl cDNA showed 38-50% identity at amino acid level among the buffalo LYZL paralogs, complete conservation of eight cysteines and other signature sequences of c-type lysozyme family. Catalytic residues in SLLP1, LYZL4 and LYZL5 have undergone replacement. The substrate binding residues showed significant variation in LYZL proteins. Residues at sites 62, 101, 114 in LYZL4; 101 in SLLP1; 37, 62, and 101 in LYZL6 were more variable among diverse species. Sites 63 and 108 occupied by tryptophan were least tolerant to variation. Site 37 also showed lower tolerance to substitution in SLLP1, LYZL4 and LYZL5, but more variable in non-testicular lysozymes. Models of LYZL proteins were created by homology modeling and the substrate binding pockets were analyzed in term of binding energies and contacting residues of LYZL proteins with tri-N-acetylglucosamine (NAG)3 in the A-B-C and B-C-D binding mode. Except LYZL6, LYZL proteins did not show significant difference in binding energies in comparison to hen egg white lysozyme in the A-B-C mode. (NAG)3 binding energy in the B-C-D mode was higher by 1.3-2.2 kcal/mol than in A-B-C mode. Structural analysis indicated that (NAG)3 was involved in making more extensive interactions including hydrogen bonding with LYZL proteins in B-C-D mode than in A-B-C mode. Despite large sequence divergence among themselves and with respect to c-type lysozymes, substrate binding residues as well as hydrogen bonding network between (NAG)3 and proteins were mostly conserved. LYZL5 in buffalo and other mammalian species contained additional 10-12 amino acid sequence at c-terminal that matched with ankyrin repeat domain-containing protein 27. Phylogenetic analysis indicated LYZL2 to be most ancient among all the LYZL proteins and that the evolution of LYZL proteins occurred through several gene duplications preceding the speciation of mammals from other vertebrates as distant as reptiles and amphibians.

Purification and characterization of CRISP-3 from human seminal plasma and its real-time binding kinetics with PSP94.

Cysteine-rich secretory proteins (CRISPs) have been postulated to have a role in male reproduction and prostate pathophysiology. Of the mammalian CRISPs, CRISP-3 levels in particular have been shown to be upregulated in prostate cancer. Efforts have been made to obtain highly pure CRISP-3 for gaining structure-function information of this protein. However, well characterized and highly pure protein is not available yet. CRISPs from snake venom have been purified using prostate secretory protein of 94 amino acids (PSP94) has been reported earlier. In the present study, CRISP-3 was purified to homogeneity from human seminal plasma using human PSP94-immnobilized affinity column. The molecular mass of the purified protein was determined by SDS-PAGE followed by immunoblotting and found to be ∼26kDa and ∼28kDa. The purity was further verified using MALDI-TOF MS analysis, where two peaks at m/z 25509 and 27715 were obtained. The lower molecular weight peak corresponds to the calculated molecular mass of CRISP-3 (∼26kDa); whereas the higher molecular weight peak was confirmed to be the glycosylated form (∼28kDa) from the deglycosylation experiment. Binding of PSP94 in increasing concentrations to purified CRISP-3 immobilized chip was further validated using surface plasmon resonance. The kinetics data suggested that purified CRISP-3 binds specifically and with high affinity to PSP94. In conclusion, a homogeneous preparation of highly pure CRISP-3 protein is obtained from human seminal plasma.

Evaluation of AMGEN clone 9G8A anti-Epo antibody for application in doping control.

The two mouse monoclonal anti-erythropoietin (EPO) antibodies clone AE7A5 (generated by using a 26 amino acid N-terminal EPO-peptide) and 9G8A (developed by immunizing mice with full length human EPO) are both directed against linear epitopes at the N-terminus of EPO. While AE7A5 has been commercially available for many years, 9G8A was made for Amgen's internal research purposes. In the past, the commercial antibody was shown to cross-react with several proteins unrelated to EPO (e.g. E. coli thioredoxin reductase, zinc-α2-glycoprotein, S. cerevisiae enolase, human neuron-specific enolase, and human non-neuronal enolase). However, it displayed high sensitivity for detecting recombinant EPO (rEPO) misuse by athletes on Western blots. We evaluated the potential use of clone 9G8A for doping control purposes. While 9G8A showed lower sensitivity than AE7A5 (ca 45% on isoelectric focusing (IEF)-polyacrylamide gel electrophoresis (PAGE), ca 40% on sodium dodecyl sulfate (SDS)- and sarcosyl (SAR)-PAGE), non-specific binding of the five proteins was not observed. The cross-reactivity of AE7A5 can be overcome by immunoaffinity purification of EPO before electrophoresis and Western blotting. Similar to AE7A5, clone 9G8A is also suited for Western double-blotting. Copyright © 2016 John Wiley & Sons, Ltd.

Sequestration of bovine seminal plasma proteins by different assemblies of phosphatidylcholine: A new technical approach.

Binder of SPerm (BSP) proteins, the main proteins from bovine seminal plasma, are known to partially intercalate into the outer leaflet of the spermatozoa membrane and bind to choline-containing lipids being present therein. This insertion generates a negative effect on semen quality after cryopreservation by inducing an early-stage capacitation of spermatozoa. The assumption of surface properties exhibited by BSP proteins was checked by tensiometry measurements: BSP proteins are highly surface active. This suggests that BSP proteins can reach the interface covered by phospholipids not only by interactions between one and each other but also due to their own surface activity. The insertion of BSP proteins into the lipid domains outer leaflet of spermatozoa was reproduced on a biomimetic system such as Langmuir monolayers. The insertion of BSP proteins can be performed in the compressible fluid domains which contain choline-bearing lipids. Monolayer films were used as well to study the complexation of BSP proteins by two phospholipid assemblies: low density lipoprotein (LDLs) from egg yolk or liposomes produced from egg phospholipids. Irrespective of the phospholipid structure (lipoprotein or liposome), BSP was hindered to alter the structure of the membrane. Only the overall ratio BSP proteins:phosphatidylcholine was important. The difference between the two sequestering agents lies on their surface properties: LDL have a strong tendency to merge with the outer layer whereas liposomes mainly remain in the bulk on the same time scale.

Sperm Lysozyme-Like Protein 1 (SLLP1), an intra-acrosomal oolemmal-binding sperm protein, reveals filamentous organization in protein crystal form.

Sperm lysozyme-like protein 1 (SLLP1) is one of the lysozyme-like proteins predominantly expressed in mammalian testes that lacks bacteriolytic activity, localizes in the sperm acrosome, and exhibits high affinity for an oolemmal receptor, SAS1B. The crystal structure of mouse SLLP1 (mSLLP1) was determined at 2.15 Å resolution. mSLLP1 monomer adopts a structural fold similar to that of chicken/mouse lysozymes retaining all four canonical disulfide bonds. mSLLP1 is distinct from c-lysozyme by substituting two essential catalytic residues (E35T/D52N), exhibiting different surface charge distribution, and by forming helical filaments approximately 75 Å in diameter with a 25 Å central pore comprised of six monomers per helix turn repeating every 33 Å. Cross-species alignment of all reported SLLP1 sequences revealed a set of invariant surface regions comprising a characteristic fingerprint uniquely identifying SLLP1 from other c-lysozyme family members. The fingerprint surface regions reside around the lips of the putative glycan-binding groove including three polar residues (Y33/E46/H113). A flexible salt bridge (E46-R61) was observed covering the glycan-binding groove. The conservation of these regions may be linked to their involvement in oolemmal protein binding. Interaction between SLLP1 monomer and its oolemmal receptor SAS1B was modeled using protein-protein docking algorithms, utilizing the SLLP1 fingerprint regions along with the SAS1B conserved surface regions. This computational model revealed complementarity between the conserved SLLP1/SAS1B interacting surfaces supporting the experimentally observed SLLP1/SAS1B interaction involved in fertilization.